Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells

Kashyap R Patel,Maria C Rodriguez Benavente,W Walter Lorenz,Emily M Mace,Adam W Barb

doi:10.1074/jbc.ra120.015516

Abstract

Many therapeutic monoclonal antibodies require binding to Fc γ receptors (FcγRs) for full effect and increasing the binding affinity increases efficacy. Preeminent among the five activating human FcγRs is FcγRIIIa/CD16a expressed by natural killer (NK) cells. CD16a is heavily processed, and recent reports indicate that the composition of the five CD16a asparagine(N)-linked carbohydrates (glycans) impacts affinity. These observations indicate that specific manipulation of CD16a N-glycan composition in CD16a-expressing effector cells including NK cells may improve treatment efficacy. However, it is unclear if modifying the expression of select genes that encode processing enzymes in CD16a-expressing effector cells is sufficient to affect N-glycan composition. We identified substantial processing differences using a glycoproteomics approach by comparing CD16a isolated from two NK cell lines, NK92 and YTS, with CD16a expressed by HEK293F cells and previous reports of CD16a from primary NK cells. Gene expression profiling by RNA-Seq and qRT-PCR revealed expression levels for glycan-modifying genes that correlated with CD16a glycan composition. These results identified a high degree of variability between the processing of the same human protein by different human cell types. N-glycan processing correlated with the expression of glycan-modifying genes and thus explained the substantial differences in CD16a processing by NK cells of different origins.

Highlights

The antibody-binding Fc γ receptors (FcγRs) capture target tissue through a linking antibody to elicit a cytotoxic cellmediated response
Of the five activating human FcγRs, FcγRIIIa/CD16a expressed by natural killer (NK) cells appears predominant for clearing cancerous cells following the administration of a therapeutic monoclonal antibody
It is notable that CD16a from NK92 cells contained a high percentage of minimally remodeled N-glycans that may result from improper protein processing during secretion

Summary

Introduction

The antibody-binding Fc γ receptors (FcγRs) capture target tissue through a linking antibody to elicit a cytotoxic cellmediated response. Our group identified multiple other surprising features on CD16a purified from primary NK cells including the presence of hybrid-type N-glycans at N45 and extensive processing including N-acetyllactosamine (LacNAc) repeats at the N38 and N74 sites (Fig. 1A). We observed striking differences in the processing at individual CD16a N-glycan sites resulting from expression with different cell types (Fig. 3).

Results

Conclusion